Electroosmosis is the movement of a liquid, relative to a stationary charge surface, which results when an electric field is applied to the liquid. Electroosmosis is related to electrophoresis, the movement of a charged surface such as that of a charged particle, relative to a stationary liquid in an electric field. Both are manifestations of surface zeta potential. The internal surfaces of the untreated glass or plastic chambers used in electrophoretic processes are characteristically charged and, under usual conditions, electroosmotic flow of the liquid near the chamber walls accompanies the desired electrophoresis of suspended sample particles or macromolecules. In the case of glass or quartz chambers, a negative charge is induced when the surface is in contact with an aqueous electrolyte medium (above the isoelectric pH of the surface). Thus, cations attracted and concentrated near the wall are characterized by a net migration toward the cathode with concomitant liquid flow due to ion solvation. This electroosmotic flow reduces the effectiveness of processes such as continuous flow electrophoresis, analytical particle electrophoresis, and isoelectric focusing (Patterson, W. J., National Aeronautics and Space Administration, Technical Memorandum, NASA TMX-73311, U.S. Government Printing Office, Washington, D.C., 1976; Shaw, D. J., "Electrophoresis", Academic Press, New York, 1969; Brinton, C. C., and Lauffer, M. A., in "Electrophoresis Theory, Methods, and Applications", (M. Bier, Ed.), Vol. 1, p. 427, Academic Press, New York, 1959; Seaman, G. V. G., in "The Red Blood Cell", (D. MasN. Surgenor, Ed.), Vol. 2, p. 1135, Academic Press, New York, 1975; Snyder, R. S., Bier, M., Griffin, R. N. Johnson, A. J., Leidheiser, H., Jr., Micale, F. J., Vanderhoff, J. W., Ross, S., and van Oss, C. M., Sep. Purif. Methods 2, 259 (1973)).
The effects of electroosmosis during electrophoresis are best observed in a small-diameter cylindrical capillary, with closed electrode ends, containing particles suspended in a dilute salt solution. As the particles migrate at a characteristic velocity per unit applied electric field strength (defined as the particle electrophoretic mobility), electroosmotic flow near the chamber wall, together with a compensating return flow of liquid down the center of the chamber, causes a parabolic distribution of particle velocities across the diameter of the cylinder. Only at the stationary level where electroosmotic fluid flow is zero, is the apparent mobility of a sample due entirely to electrophoresis. Thus, electroosmosis limits automation of analytical particle electrophoresis, since observations must be made while focus is maintained precisely at the stationary level. In addition, electroosmosis hinders preparative electrophoretic separations and the use of more optically suitable electrophoresis chambers of rectangular cross-sectional shape.
If a capillary possessing uniform surface charge density is filled with a dilute suspension of particles of uniform surface charge density, the electrophoretic behavior of liquid and particles can be defined mathematically (Shaw, D. J., "Electrophoresis", Academic Press, New York, 1969; Brinton, C. C., and Lauffer, M. A., in "Electrophoresis Theory, Methods, and Applications", (M. Bier, Ed.), Vol. 1, p. 427, Academic Press, New York, 1959). In such cases the stationary level is located a distance from the capillary wall equal to 0.146 of the inner diameter, and the electroosmotic mobility (at the capillary wall) is equal to the negative (indicating direction) of the difference between the observed particle mobility along the center axis of the capillary and the particle mobility at the stationary level. (Such a level is more difficult to define in rectangularly shaped chambers.) At any given location across the diameter of a capillary, the net particle mobility is equal to the sum of the particle electrophoretic mobility and the fluid mobility due to electroosmosis.
In the absence of appreciable gravity, disruptive convective fluid movement, due to the temperature and concentration gradients inherent in electrophoresis, is eliminated. Consequently, electrophoresis in space has been a major part of the National Aeronautics and Space Administration (NASA) materials processing experiments since the early 19703 s (Synder, R. S., Bier, M., Griffin, R. N., Johnson, A. J., Leidheiser, H., Jr., Micale, F. J., Vanderhoff, J. W., Ross, S., and van Oss, C. M., Sep. Purif. Methods 2, 259 (1973). Snyder, R. S., in "Electrophoresis 81" (R. C. Allen and P. Arnaud, Ed.), p. 883, Walter de Gruyter, New York, 1981). However, electroosmotic fluid flow does occur in microgravity, decreasing the resolution of electrophoretic separations (Snyder, R. S., Bier, M., Griffin, R. N., Johnson, A. J., Leidheiser, H., Jr., Micale, F. J., Vanderhoff, J. W., Ross, S., and van Oss, C. M., Sep. Purif. Methods 2, 259 (1973); Hjerten, S., Chromatog. Rev., 9, 122 (1967)).
Previous attempts to control this fluid flow have involved use of methylcellulose with molecular weight (MW) 110 000 as a wall coating, (Patterson, W. J., National Aeronautics and Space Administration, Technical Memorandum, NASA TMX-73311, U.S. Government Printing Office, Washington, D.C., 1976; Hjerten, S., Chromatog. Rev. 9, 122 (1967); Micale, F. J., Vanderhoff, J. W., and Snyder, R. S., Sep. Purif. Methods, 5, 361 (1976); Allen, R. E., Rhodes, P. H., Snyder, R. S., Barlow, G. H., Bier, M., Bigazzi, P. E., van Oss, C. J., Knox, R. J., Seaman, G. V. F., Micale, F. J., and Vanderhoff, J. W., Sep. Purif. Methods, 6, 1 (1977)). Not only does methylcellulose possess very few charged groups, but the long polymer chains appear to extend the plane of shear sufficiently beyond the double layer (Debye length) to result in near-zero zeta potential (Patterson, W. J., National Aeronautics and Space Administration, Technical Memorandum, NASA TMX-73311, U.S. Government Printing Office, Washington, D.C., 1976; Shaw, D. J., "Electrophoresis", Academic Press, New York, 1969; Brinton, C. C., and Lauffer, M. A., in "Electrophoresis Theory, Methods, and Applications" (M. Bier, Ed.), Vol. 1, p. 427, Academic Press, New York, 1959; Seaman, G. V. F., in "The Red Blood Cell" (D. MacN. Surgenor, Ed.), Vol. 2, p. 1135, Academic Press, New York, 1975). Methylcellulose coatings virtually eliminate electroosmosis. However, even when attachment of methylcellulose to glass electrophoresis chamber surfaces is enhanced by prior treatment of the surfaces with the silane, A-glycidoxypropyltrimethoxysilane, the methylcellulose coatings readily desorb during storage (Patterson, W. J., National Aeronautics and Space Administration, Technical Memorandum, NASA TMX-73311, U.S. Government Printing Office, Washington, D.C., 1976; Micale, F. J., Vanderhoff, J. W., Sep. Purif. Methods, 6, 1 (1977); Allen, R. E., Rohdes, P. H., Snyder, R. S., Barlow, G. H., Bier, M., Bigazzi, P. E., van Oss, C. J., Knox, R. J., Seaman, G. V. F., Micale, F. J., and Vanderhoff. J. W., Sep. Purif. Methods, 6, 1 (1977); Ma, S. M., Gregonis, D. E., van Wagenen, R. A., and Andrade, J. D., in "Hydrogels for Medical and Related Applications" (J. D. Andrade, Ed.), Amer. Chem. Soc. Symp. Series, Vol. 31, p. 241, 1976). In such instances, methylcellulose desorption can be detected through the measurement of electrophoretic mobilities of suspended polystyrene latex (PSL) spheres, which strongly adsorb free methylcellulose from the medium. This adsorption, which results in an easily measureable reduction in PSL particle electrophoretic mobility, appears to be of high affinity and fairly stable under test conditions (Patterson, W. J., National Aeronautics Space and Administration, Technical Memorandum, NASA TMX-73311, U.S. Government Printing Office, Washington, D.C., 1976).
The problem of methylcellulose coating instability has led to the investigation of other polymer coatings for electroosmosis control and to the development of methods for evaluation of the coatings. Measurement of streaming potential is one method that has been used to evaluate the effectiveness of coatings (Ma, S. M., Gregonis, D. E., van Wagenen, R. A., and Andrade, J. D., in "Hydrogels for Medical and Related Applications" (J. D. Andrade, Ed.), Amer. Chem. Soc. Symp. Series, Vol. 31, p. 241, 1976; van Wagenen, R. A., and Andrade, J. D., J. Colloid Interface Sci. Vol. 76, No. 2, p. 305 (1980); van Wagenen, R. A., Coleman, D. L., King, R. N., Triolo, P., Brostrom, L., Smith, L. M., Gregonis, D. E., and Andrade, J. D., J. Colloid Interface Sci., 84, 155 (1981)).
In Ma et al., hydrophilic polymer coatings apparently covalently bound to glass were used, including methylcellulose; hydroxypropylmethylcellulose; dextrin; agarose; hydroxyethylmethacrylate; methoxyethylmethacrylate; and methoxyethoxyethylmethacrylate. In a later publication by this group, the effect of various neutral and charged biopolymer and synthetic polymer thin films on electroosmosis was studied. The thin films were supported on glass or vinylsilanized glass substrates. Part of the films might have been covalently bonded to the glass, especially in the case of methacrylates. However, many of the films were adsorbed onto the glass substrate. These films were thick relative to the monomolecular coatings of this invention.
van Wagenen et al (1980) note that there are advantages to using flat plates rather than capillary tubes to accurately measure streaming potential.
In general, the silyl coatings reduce surface zeta potential by more than 30%; they are probably more useful as sublayer coatings to enhance the absorption or covalent coating by polymers (Patterson, W. J., National Aeronautics and Space Administration, Technical Memorandum, NASA TMX-73311, U.S. Government Printing Office, Washington, D.C., 1976; Hannig, K., Wirth, H., Meyer, B. H., and Zeiller, K., Hoppe Seyler's Z. Physiol. Chem., 356, 1209 (1975); Nordt, F. J., Knox, R. J., and Seaman, G. V. F., in "Hydrogels for Medical and Related Applications" (J. D. Andrade, Ed.), ACS Symposium Series, No. 31, Amer. Chem. Soc., 1976; Menawat, A., Henry, J., Jr., and Siriwardane, R., J. Colloid Interface Sci., 101 (1984); Lee, L. H., J. Colloid Interface Sci., 27, 751 (1968)). Electrophoresis chambers have been coated with carbohydrates such as agarose (van Wagenen, R. A., Coleman, D. L., King, R. N., Triolo, P., Brostrom, L., Smith, L. M., Gregonis, D. E., and Andrade, J. D., J. Colloid Interface Sci., 84 155 (1981); Nordt, F. J., Knox, R. J., and Seaman, G. V. F., in "Hydrogels for Medical and Related Applications" (J. D. Andrade, Ed.), ACS Symposium Series, No. 31, Amer. Chem. Soc., 1976; Ragetli, H. W. J., and Weintraub, M., Biochem. Biophys. Acta., 112, 160 (1966); Porath, J., Johnson, J., and Laas, T., J. Chromatog., 60, 167 (1971); van Oss, C. J., Fike, R. M., Good, F. J., and Reinig, J. M., Anal. Biochem., 60, 242 (1974) and dextran, (Nordt, F. J., Knox, R. J., and Seaman, G. V. F., in "Hydrogels for Medical and Related Applications" (J. D. Andrade, Ed.), ACS Symposium Series, No. 31, Amer. Chem. Soc., 1976) with much the same success as for methyl-cellulose. van Wagenen et al used streaming potential measurements to investigate the ability of a number of coatings to control glass and plastic zeta potentials (Ma, S. M., Gregonis, D. E., van Wagenen, R. A., and Andrade, J. D., in "Hydrogels for Medical and Related Applications" (J. D. Andrade, Ed.), Amer. Chem. Soc. Symp. Series, Vol. 31, p. 241, 1976; van Wagenen, R. A., and Andrade, J. D., J. Colloid Interface Sci., Vol. 76, No. 2, p. 305 (1980); van Wagenen, R. A., Coleman, D. L., King, R. N., Triolo, P., Brostrom, L., Smith, L. M., Gregonis, D. E., and Andrade, J. D., J. Colloid Interface Sci., 84 155 (1981)). Their results indicate that suitable coatings may be methacrylate hydrogels with ionogenic groups incorporated into the polymer. These coatings are difficult to apply, but they are capable of increasing, decreasing, or changing the sign of the streaming potentials. One drawback is that the coating charge would have to be balanced to the native surface charge at a particular pH, thus limiting their usefulness especially in the case of isoelectric focusing (where a pH gradient is utilized). Also the charged polymeric coatings would be expected to exhibit greater adsorption of biological material (i.e., proteins) than neutral hydrophilic polymeric coatings. A different method for evaluation of coatings was used by Hannig, K., Wirth, H., Meyer, B. H., and Zeiller, K., Hoppe Seyler's Z. Physiol. Chem., 356, 1209 (1975). They evaluated the effectiveness of several coatings used to coat chambers in free flow electrophoretic separations (C.F.E.) by comparing the degrees of sample band resolution. This reference discloses that high molecular weight polyethers (e.g., PEG; MW. 200,000 to 600,000) were applied to a glass substrate. However, no mention of the method of application of the polyethers is set forth in this reference, but presumably a chromatography method was used which involves adsorption of the polyethers to glass rather than covalent binding. Further, the authors were attempting to find a method or apparatus to control electroosmosis [as a controlled amount of electroosmosis is desirable in C.F.E.], but failed. Rather, all signs of electroosmosis were eliminated.